Monoclonal antibodies, antigens and diagnosis and therapy of malignant diseases

ABSTRACT

The invention concerns novel DNA and amino acid sequences of monoclonal antibodies (mAbs) raised against lymphoblastoid cells and peptides to which the mAbs bind to. The invention also concerns diagnostic assays using said antibodies or peptides for detecting individuals with a high probability of having a malignant disease and, at times, for detecting an individual having a specific malignant disease. The invention further concerns pharmaceutical compositions comprising the mAbs or peptides of the invention for use in the treatment of various malignant diseases as well as methods for the treatment of malignant diseases using the mAbs or peptides of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of the U.S. National Stagedesignation of International Application PCT/IL99/00518 filed Sep. 30,1999, the content of which is expressly incorporated herein by referencethereto.

FIELD OF THE INVENTION

[0002] The present invention concerns novel sequences of monoclonalantibodies, peptidic sequences of antigens to which the monoclonalantibodies bind, as well as diagnostic and therapeutic assays using themonoclonal antibody and peptides.

BACKGROUND OF THE INVENTION

[0003] Co-owned PCT Application, Publication No. WO 95/20605, disclosesimmuno-stimulatory monoclonal antibodies. The antibodies subject of thisPCT application were raised against B lymphoblastoid cells and wereshown to have an immuno-stimulatory effect. When injected intotumor-bearing animals, these antibodies were also found to elicit ananti-tumor effect.

[0004] Cancer diagnosis, under current medical procedures, is typicallya multi-step process involving physical examination, use of a variety ofimaging techniques, employment of a variety of cancer markers, etc.There is a longfelt need in the art for cancer diagnostic techniqueswhich allow detection of cancer and also determination of the type ofcancer which the tested individual is suffering from.

GENERAL DESCRIPTION OF THE INVENTION

[0005] The present invention is based on the finding of sequences ofmonoclonal antibodies against lymphoblastoid cells. The presentinvention is further based on the finding that the level of binding ofthese antibodies to T-cells of patients having cancer is different(higher or lower) than the level of binding of these antibodies toT-cells of healthy individuals.

[0006] In accordance with one aspect of the invention there is provideda monoclonal antibody having a variable region selected from the groupconsisting of:

[0007] (a) a monoclonal antibody having a heavy chain variable regioncomprising the amino acid sequence of FIG. 1;

[0008] (b) a monoclonal antibody having a Kappa light chain variableregion comprising the amino acid sequence of FIG. 2;

[0009] (c) a monoclonal antibody having a heavy chain variable regioncomprising the amino acid sequence of FIG. 1 and the Kappa light chainvariable region comprising the amino acid sequence of FIG. 2;

[0010] (d) a monoclonal antibody having a heavy chain variable regionhaving at least 70% identity to the amino acid sequence of FIG. 1;

[0011] (e) a monoclonal antibody having a light chain variable regionhaving at least 70% identity to the sequence of FIG. 2.

[0012] In accordance with the invention, the term “antibody” refers tomonoclonal antibodies of any of the classes IgG, IgM, IgD, IgA and IgE.The term refers to whole antibodies or fragments of the antibodiescomprising the antigen-binding domain of the antibodies, e.g. antibodieslacking the Fc portion, single chain antibodies, fragments of articlesconsisting essentially of only the variable antigen-binding domain ofthe antibody, etc.

[0013] In addition the invention also concerns antibodies which bind toan antigen to which any one of the above mAbs specifically binds to i.e.antibodies which have cross reactivity with the above antibodies.

[0014] In accordance with one embodiment of the invention, themonoclonal antibody is a chimeric human-mouse antibody, namely a mAbwith a constant region derived from a human origin and a variable regionderived from mouse. For this purpose, the Kappa light and heavy chainvariable regions of the mAb of the invention were PCR cloned and theirDNA sequenced. In accordance with yet another embodiment of theinvention the antibody is a fully humanized antibody, i.e. both itsvariable and constant region are derived from a human source.

[0015] The term “having at least X percent identity” refers to thepercent of amino acid residues that are identical in the two comparedsequences when the sequences are optimally aligned. Thus, 70% amino acidsequence identity means that 70% of the amino acids in two or moreoptimally aligned polypeptide sequences are identical. Preferably, theidentity is at least 80%, most preferably at least 90%.

[0016] In accordance with an additional aspect of the invention, thereare provided mouse hybridoma cell lines which produce any of the mAbs ofthe invention. The hybridomas may be prepared by any of the methodsknown in the art (for example, Kohler, G. and Milstein, C., Nature,256:495-497, (1975)). The supernatant of the hybridoma cell lines aretypically screened for antibody binding activity by any one of themethods known in the art such as by enzyme linked immuno sorbent assay(ELISA) or radio immuno assay (RIA). The supernatants are screened forproduction of mAbs which bind to any of the peptides of the invention(as explained below) or which bind to cells to which they bind, e.g.Daudi cells or T lymphocytes.

[0017] DNA sequences which encode any of the amino acid sequences of theheavy chain or light chain of the above mAbs are also encompassed withinthe scope of the invention. As will no doubt be clear to any man versedin the art, due to the degenerative nature of the genetic code aplurality of nucleic acid sequences may code for the mAb of theinvention beyond those shown in FIGS. 1 or 2.

[0018] The invention also provides expression vectors such as plasmidshaving said DNA sequences as well as host cells containing one or moreof these expression vectors.

[0019] In accordance with another aspect of the invention, there areprovided peptidic sequences of a B-cell antigens to which the mAbs ofthe invention can bind. Searches performed against the non-redundantgene bank database and the EST division determined that these peptidicsequences are novel.

[0020] In accordance with this additional aspect of the invention thereis provided a peptide selected from the group consisting of:

[0021] (a) a peptide having an amino acid sequence as depicted in FIG.10;

[0022] (b) a peptide having an amino acid sequence as depicted in FIG.11;

[0023] (c) a peptide having an amino acid sequence as depicted in FIG.12;

[0024] (d) a peptide having at least 85% identity to any one of theamino acid sequences of the peptides of (a), (b) and (c) above; and

[0025] (e) a protein or a peptide comprising one or more of the peptidesof (a)-(d) above.

[0026] The peptides of the invention may be used for a variety ofdiagnostic assays, such as, for example, competitive immuno-assayswherein the level of binding of the mAb of the invention to its nativeantigen, which exists on T-cells is determined. In addition, thepeptides may be used for the production of antibodies in immunizedanimals which antibodies may then be used for any one of the utilitiesdescribed above and below.

[0027] Analogs of all the above peptides also form an additional aspectof the present invention. As will be appreciated by an person versed inthe art, the amino acid sequence of the peptides of the invention may bealtered, for example, by addition, deletion or conservative ornon-conservative substitution of one or more amino acids withoutsubstantially altering the antibody binding properties of the peptide

[0028] The term “conservative substitution” refers to the substitutionof an amino acid in one class by an amino acid of the same class, wherea class if defined by common physiochemical amino acid side chainproperties and high substitution frequencies in homologous proteinsfound in nature, as determined, for example, by a standard Dayhofffrequency exchange matrix or BLOSUM matrix. [Six general classes ofamino acid side chains have been characterized and include: Class I(Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln,Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and ClassVI (Phe, Tyr, Trp). For example, substitution of an Asp for anotherClass III residue such as Asn, Gln, or Glu, is a conservativesubstitution. The term “non-conservative substitution” refers to thesubstitution of an amino acid in one class with an amino acid fromanother class; for example, substitution of an Ala, a Class II residue,with a Class III residue such as Asp, Asn, Glu, or Gln.

[0029] The letters used above (and hereinafter) to denote specific aminoacids (aa) are in accordance with the 1-letter amino acid symbolsrecommend by the IUPAC-IUB Biochemical Nomenclature Commission.

[0030] Analogs of the above peptides which fall under the scope of thepresent invention are such which have substantially the same level ofbinding to the mAbs of the invention as the peptides depicted in FIGS.10-12. The level of binding can be determined by any manner known in theart.

[0031] The peptides and analogs of the invention may also be chemicallymodified and such chemically modified peptides and analogues also form apart of the invention. The term “chemically modified” refers to aprotein where at least one of its amino acid residues is modified eitherby natural processes, such as processing or other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Among the numerous known modifications typical, butnot exclusive examples include: acetylation, acylation, amidation,ADP-ribosylation, glycosylation, GPI anchor formation, covalentattachment of a liquid or lipid derivative, methylation, myristylation,pegylation, prenylation, phosphorylation, ubiqutination, or any similarprocess.

[0032] The second finding on which the invention is based is that themAbs of the invention can bind to a different extent to T-cells obtainedfrom individuals having a malignant disease as compared to the extent ofbinding of the same mAbs to T-cells of a healthy individual.

[0033] Thus, by a further aspect of the present invention an assay isprovided for identifying a tested individual with a high probability ofhaving a malignant disease comprising:

[0034] (a) obtaining a body fluid sample from said individual;

[0035] (b) contacting said sample with at least one mAb of theinvention;

[0036] (c) determining the extent of binding of said mAbs to T-cellswithin said sample; and

[0037] (d) comparing the extent of (c) to the extent of binding of themAbs of the invention to T-cells in a sample obtained from a healthyindividual; a significant difference between the above two extents ofbinding indicating that said tested individual has a high probability ofhaving a malignant disease.

[0038] In accordance with the invention, the sample obtained from theindividual to be tested may be any body fluid which contains adetectable amount of T-cells. Typically, the body fluid sample is ablood or lymph fluid sample. Preferably, before contacting the mAbs ofthe invention with the obtained sample, the peripheral blood monoclearcells (PBMC) in the sample are separated by any one of the methods knownin the art such as by Ficoll Hypaque density centrifugation and theseparated cells are then contacted with the tested antibodies.

[0039] The term “malignant disease” in accordance with the invention isto be understood as any kind of malignant disease known in the art atany of its stages.

[0040] This term also encompasses malignant diseases which are at theirearly stages and have not yet elicited clinical symptoms. Preferablythis term refers to solid tumors.

[0041] The term “healthy individual” relates to an individual who doesnot have a malignant disease, and may also refer to an average level ofseveral individuals or to a level obtained by pooling together bodyfluids from several individuals. It should be noted that once a standardextent of binding of healthy individuals is established, there is noneed to re-establish this standard for every test and the figureestablished may be used continuously. In accordance with the inventionit has been found that in healthy individuals about 25% of CD3⁺ T-cellsbind to antibodies of the invention.

[0042] The term “high probability” means that the assay of the inventionis an initial screening assay capable of identifying individualssuspected of having a malignant disease. The fact that the individualdetected by the method of the invention has indeed a malignant diseasewill have to be verified later by utilizing additional techniques knownin the art.

[0043] The term “extent of binding” relates to the level of binding ofthe antibody to an antigen present on the T-cell of the testedindividual which extent can be determined by any of the methods known inthe art for determining binding levels of antibodies such as ELISA orWestern Blotting. The extent of binding may be determined using anydetection system such as anti-mouse immunoglobulin or fragments thereoflinked to a detectable marker. Examples of such detectable markers are aradioactive group, a fluorescent group, an enzyme capable of catalyzinga reaction yielding a detectable product (such as a color reaction), abiotin group capable of being detected by avidin, etc. By a preferredembodiment, the extent of binding of the mAbs of the invention to theT-cells is carried out by double labeling in which the anti T-cellantibody (e.g. anti-CD3⁺ antibody) is attached to one kind offluorescent marker and the mAb of the invention is attached to a secondtype of fluorescent marker. The extent of binding is then determinedusing fluorecein activated cell sorter (FACS). The quantitation of theextent of binding is achieved by determining the percent of CD3⁺ T-cells(determined by their binding of anti-CD3⁺ antibodies) which also bindthe mAb of the invention.

[0044] In accordance with the invention, it was found that the totalnumber of CD3⁺ cells in blood samples of individuals having a malignantdisease is similar to the number of CD3⁺ cells in blood samples obtainedfrom healthy individuals so that the normalization of the extent ofbinding of both mAb and CD3⁺ T-cells by using total CD3⁺ binding T-cellsboth in malignant patients and healthy individuals is valid. However,the percent of the CD3⁺ binding T-cells which also bind the mAb of theinvention (hereinafter: “CD3⁺ mAb cells”) in individuals having amalignant disease differs significantly from the percent of CD3⁺ mAb⁺cells in blood of healthy individuals. The percent of the CD3⁺ mAb⁺cells in an individual having a malignant disease may either besignificantly higher or significantly lower than the percent of CD3⁺mAb⁺ cells in healthy individuals, depending on the type of themalignant disease.

[0045] The extent of binding of a mAb of the invention to a T-cellobtained from a tested individual will be considered to be“significantly different” than the extent of binding to T-cells obtainedfrom a healthy individual when the difference in binding of the mAb isstatistically different in a significant degree as determined by any ofthe statistical methods known in the art (e.g. Students t-Test) whichare used in connection with results obtained by the experimental methodsmentioned herewith.

[0046] The invention not only enables to identify individuals having ahigh probability of having any type of malignant diseases (where thediseased individual has a different extent of binding of T-cells to mAbsof the invention as compared to a healthy individual) but can also helpidentify individuals having specific types of cancer by determiningwhether said extent is higher or lower than the corresponding extent inthe healthy individual.

[0047] Typically, the percent of binding of the mAbs of the invention toT-cells obtained from healthy individuals is in the range of about 25%,i.e. 25% of the cells expressing the CD3⁺ T-cell marker (determined bybinding of anti-CD3⁺ antibody to the cells) also bind the mAbs of theinvention.

[0048] In accordance with the invention, it has been shown that insamples obtained from prostate cancer patients, the percent of CD3⁺T-cells to which the mAbs of the invention bind are in the range ofabout 50%.

[0049] It was further shown that where the CD3⁺ T-cells originate fromsamples obtained from colon or breast carcinoma patients, the percent ofthe cells which also bind to the mAbs of the invention is about 7% and10%, respectively.

[0050] Thus, in accordance with the present invention it has becomepossible to determine that there is a high probability that there existsa specific type of cancer in a body fluid sample taken from a testedindividual using a simple and single assay based on the extent ofbinding of the mAbs of the invention to CD3⁺ cells present in the bodyfluid sample. The simplicity of the diagnostic assay of the inventionwhich necessitates use of only one kind of mAb to identify an individualhaving a certain type of cancer is very useful for wide screening of apopulation.

[0051] Thus, the present invention by another of its aspects provides anassay for identifying a tested individual with a high probability ofhaving a specific malignant disease comprising:

[0052] (a) obtaining a body fluid sample from said individual;

[0053] (b) contacting said sample with the mAbs of the invention;

[0054] (c) determining the extent of binding of said mAbs to T-cells insaid sample; and

[0055] (d) comparing the extent of binding (c) cells obtained to theextent of binding of the mAbs to T-cells obtained from a healthyindividual, the existence of a significant difference in the extents ofbinding indicating with a high probability that the tested individualhas a malignant disease wherein whether the extent of binding to theT-cells from said individual is above or below the extent of the bindingof the mAbs in T-cells of healthy individuals, indicates a specific typeof malignant disease which the tested individual has with highprobability.

[0056] In particular, where the extent of binding to the mAb of theinvention is significantly higher than in healthy individuals the testedindividual has a high probability of having prostate cancer.

[0057] Where the extent of binding is significantly lower than thehealthy individual, the tested individual has a high probability ofhaving colon or breast cancer.

[0058] In accordance with the diagnostic aspect of the invention,compositions comprising the mAbs of the invention may be used fordiagnosis to identify individuals with the high probability of having amalignant disease (in general) or for identifying a specific malignantdisease the individual is likely to have. The invention thereforeprovides by another of its aspects, a diagnostic composition comprisingmAbs belonging to at least one of the abovementioned antibodies togetherwith a suitable carrier. The carrier may either be a soluble carriersuch as any one of the physiological acceptable buffers known in the art(e.g. PBS) or a solid state carrier such as, for example, latex beads.

[0059] The present invention also provides kits, e.g. diagnostic assaykits, for utilizing the mAbs of the invention and carrying out thediagnostic assays disclosed above. In one embodiment, the diagnostic kitwould conventionally include at least one of the above mAbs in one ormore containers, a conjugate of a specific binding partner for the mAb(for example the antigen or analog of the invention), a label capable ofproducing a detectable signal and directions for its use. The label maybe, a priori, bound to the monoclonal antibody or, alternatively, thelabel may be bound to a carrier molecule which then specifically bindsto the mAb. The incubation of the tested sample with the diagnosticreagent composition is for a time sufficient to allow binding of themonoclonal antibodies to the cells.

[0060] By a further aspect of the invention, there are providedpharmaceutical compositions comprising, as an active ingredient, one ormore of the mAbs of the invention together. Use of said mAbs for thepreparation of pharmaceutical preparations for the treatment of variousmalignant diseases in an individual is also within the scope of theinvention.

[0061] By yet another aspect the present invention concerns a method oftreatment of malignant diseases by administering to an individual inneed a therapeutically effective amount of said mAbs. A therapeuticallyeffective amount being an amount capable of alleviating the symptoms ofthe malignant disease, reducing the symptoms or completely eliminatingthem.

[0062] Pharmaceutical compositions comprising the peptides of theinvention also constitute an aspect of the invention. Such compositionsmay be used, for example, for active immunization of an individual toobtain antibodies which may then bind to the T-cells of the individualand elicit an immune response in the individual.

DETAILED DESCRIPTION OF THE ASPECTS OF THE INVENTION

[0063] The main aspects of the invention will now be described withoccasional reference to the attached figures. In the followingdescription and figures, the term “BAT antibody” will be usedinterchangeably with the term “mAbs of the invention”.

BRIEF DESCRIPTION OF THE FIGURES

[0064]FIG. 1 shows the DNA (SEQ ID NO.: 1) and peptide sequences (SEQ IDNO.: 2) of the heavy chain variable region of the mAb of the invention;.

[0065]FIG. 2 shows DNA (SEQ ID NO.: 3) and peptide sequences (SEQ IDNO.: 4) of the Kappa light chain variable region of the mAb of theinvention;

[0066]FIG. 3 shows an analysis of the amino acid sequence of the heavychain variable region of the antibody of the invention (designated “BAT“BAT” defines the amino acid sequence of the BAT antibody V_(H) region,while “VMS2” defines the amino acid sequence of the germline VMS2/VGK4germline gene. Where the BAT sequence and the germline sequence areidentical the germline sequence is represented by a dot (.); wheremismatches occur the different germline residue is shown. The tablesbelow, the sequence on the following pages describe the frequency withwhich certain amino acids have been seen at a particular residueposition both within the Kabat et al., Sequences of proteins ofimmunological interest, (1991) mouse heavy chain subgroup miscellaneous(Mouse V_(H) Misc.) and across a larger database of all known mouseV_(H) sequences (All Mouse V_(H));.

[0067]FIG. 4 shows an analysis of the amino acid sequence of the kappalight chain variable region of the antibody of the invention (designatedin the FIG. As “BAT”). “Mouse” defines the amino acid sequence of theBAT antibody K_(K) region, while “Germ” defines the amino acid sequenceof the germline H4 germline gene. Where the BAT sequence and thegermline sequence are identical the germline sequence is represented bya dot (.); where mismatches occur the different germline residue isshown. The tables below and on the following pages describe thefrequency with which certain amino acids have been seen at a particularresidue position both within the Kabat mouse heavy chain subgroup VI(Mouse V_(K) VI) and across a larger database of all known mouse V_(K)sequences (All Mouse V_(K));

[0068]FIG. 5 shows the DNA (SEQ ID NO.: 5) and peptide sequences (SEQ IDNO.: 6) of the Kappa light chain variable regions of the chimericantibody of the invention;

[0069]FIG. 6 shows the DNA (SEQ ID NO.: 7) and peptide sequences (SEQ IDNO.: 8) of the heavy chain variable region of the chimeric antibody ofthe invention;

[0070]FIG. 7 shows a schematic representation of the pKN 110 mammalianexpression vector used for the expression of the Kappa light chain ofthe chimeric antibody of the invention;

[0071]FIG. 8 shows a schema tic representation of the pG1D 110 mammalianexpression vector used for the expression of the heavy chain of thechimeric antibody of the invention.

[0072]FIG. 9 shows a graphic representation featuring an example ofresults of an ELISA assay measuring the binding characteristics of themouse and the γ1/Kappa chimeric antibody of the invention to Daudicells;.

[0073]FIG. 10 shows the amino acid sequence of peptide 1 (SEQ ID NO.: 9)of the invention;

[0074]FIG. 11 shows the amino acid sequence of peptide 2 (SEQ ID NO.:10) of the invention;

[0075]FIG. 12 shows the amino acid sequence of peptide 3 (SEQ ID NO.:11) of the invention;

[0076]FIG. 13 is a schematical representation showing the percent ofCD3⁺ cells which also bind the mAb of the invention (indicated as “BAT”)as compared to the total number of CD3⁺ cells in blood samples ofhealthy individuals as determined by FACS analysis;

[0077]FIG. 14 shows the percent of CD3⁺ cells which also bind the mAb ofthe invention (indicated as BAT) as compared to the total number of CD3⁺cells in blood samples taken from patients having colon carcinoma asdetermined by FACS analysis;

[0078]FIG. 15 shows the percent of CD3⁺ cells which also bind the mAb ofthe invention (indicated as BAT) as compared to the total number of CD3⁺cells in blood samples obtained from patients having breast carcinoma;

[0079]FIG. 16 shows the percent of CD3⁺ cells which also bind the mAb ofthe invention (indicated as BAT) as compared to the total number of CD3⁺cells in blood samples obtained from patients having prostate carcinoma;

[0080]FIG. 17 is a schematic representation showing the mean percent ofCD3⁺ cells which bind the mAb of the invention (indicated as BAT) inhealthy individuals as compared to patients having breast carcinoma,colon carcinoma or prostate carcinoma;

[0081]FIG. 18 is a photograph of a Western Blot of peptides obtainedfrom T-cells of individuals having prostate cancer, ear, nose and throat(ENT) carcinoma, breast carcinoma or from membranes of Daudi cells. TheBlot was incubated with the mAb of the invention and shows an increasedamount of antigen in T-cells obtained from patients having prostatecarcinoma as compared to an undetectable level of antigen in T-cellsobtained from patients having breast carcinoma;

I. SEQUENCING OF THE MAB

[0082] (A) Abbreviations

[0083] Fetal Calf Serum (FCS); ribonucleic acid (RNA); messenger RNA(mRNA); deoxyribonucleic acid (DNA); copy DNA (cDNA) ; polymerase chainreaction (PCR); minute (min); second (sec); Tris-borate buffer (TBE).

[0084] (B) Materials

[0085] Media components and all other tissue culture materials wereobtained from Life Technologies (UK). The RNA isolation kit was obtainedfrom Stratagene (USA) while the 1^(st) strand cDNA synthesis kit waspurchased from Pharmacia (UK). All the constituents and equipment forthe PCR-reactions, including AmpliTaq® DNA polymerase, were purchasedfrom Perkin Elmer (USA). The TA Cloning® kit was obtained fromInvitrogen (USA). Agarose (UltraPure™) was obtained from LifeTechnologies (UK). The Thermo Sequences™ pre-mixed cycle sequencing kitand the Vistra 725 DNA sequencing machine were both purchased fromAmersham (UK). All other molecular biological products were obtainedfrom New England Biolabs (USA).

[0086] (C) Experimental Techniques:PCR Cloning and Sequencing of theMouse BAT Antibody Variable Region Genes

[0087] The mouse BAT hybridoma cell line and the Daudi cell line weresuccessfully transferred to the MRC-CC and both cell lines were grown,in suspension, using RPMI (without glutamine) supplemented with 10%(v/v) FCS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mML-glutamine, 1 mM sodium pyruvate and 12.5 units/ml Nystatin.

[0088] Approximately 10⁸ of viable cells of the BAT hybridoma cell linewere harvested and, from the 10⁸ cells, total RNA was isolated using anRNA Isolation kit according to the manufacturers instructions. The kitused a guanidinium thiocyanate phenol-chloroform single step extractionprocedure as described by Chromczynski and Sacchi, Anal. Biochem.,162:156, 1987. Also following the manufacturers instructions a 1^(st)Strand cDNA synthesis kit was employed to produce a single-stranded DNAcopy of the BAT hybridoma mRNA using the NotI-(dT)₁₈ primer supplied inthe kit. Approximately 5 μg of total RNA was used in each 33 μl finalreaction volume. The completed reaction mix was then heated to 90° C.for 5 min. to denature the RNA-cDNA duplex and inactivate the reversetranscriptase, before being chilled on ice.

[0089] To PCR-amplify the mouse heavy chain variable region gene (V_(H)gene) and the mouse kappa light chain variable region gene (V_(K) gene)from the hybridoma cell line the method described by Jones and Bendig,Bio/Technology, 9:8, 1987 was followed. Essentially, two series ofdegenerate primers, one designed to anneal to the leader sequences ofthe mouse heavy chain genes (i.e. MHV1-12; Table 1) and one designed toanneal to the leader sequences of mouse kappa light chain genes (i.e.MKV1-11; Table 2) were used, in conjunction with primers designed toanneal to the 5′-end of the appropriate constant region gene, toPCR-clone the murine variable region genes.

[0090] Separate PCR-reactions were prepared for each of the degenerateprimers with their appropriate constant region primer, in a specialPCR-room using specific protocols designed to minimize the possibilityof cross-contamination. Amplitaq® DNA polymerase was used to amplify thetemplate cDNA in all cases. The PCR-reaction tubes were than loaded intoa Perkin Elmer 480 DNA thermal cycler and cycled (after an initial meltat 94° C. for 1.5 min) at 94° C. for 1 min and 72° C. for 1 min over 25cycles. At the completion of the last cycle a final extension step at72° C. for 10 min was carried out before the reactions were cooled to 4°C. Except for between the annealing (50° C.) and extension (72° C.)steps, when an extended ramp time of 2.5 min was used, a 30 sec ramptime between each step of the cycle was employed.

[0091] 10 μl aliquots from each PCR-reaction were run on a 1%agarose/TBE (pH 8.8) gel to determine which had produced a PCR-productof the correct size. Those PCR-reactions that did appear to amplifyfull-length variable region genes were repeated to produce independentPCR-clones and thereby minimize the effect of PCR-errors. 1-6 μ1aliquots of those PCR-products of the correct size were directly clonedinto the pCRII™ vector, provided by the TA Cloning® kit, and transformedinto INA αF′ competent cells as described in the manufacturersinstructions. Colonies containing the plasmid, with a correctly sizedinsert, were identified by PCR-screening the colonies using the pCRIIForward and pCRII Reverse oliognucleotide primers described in Table 3below according to the method of Güssow and Clackson, Nucleic AcidsRes., 17:4000, 1989

[0092] Those putative positive clones identified were double-strandedplasmid DNA sequenced using the Vistra DNA sequencing machine and theThermo Sequenase™ pre-mixed cycle sequencing kit as described in themanufacturers instructions.

EXAMPLE 1 Cloning and Sequencing of the Heavy Chain Variable Region ofthe BAT Antibody

[0093] As with all humanization projects, a strict PCT-cloning andsequencing protocol was followed. This was done to minimize thepossibility of introducing errors into the wild-type sequences of themouse VH variable region genes from the BAT hybridoma cell line. Only ifall the DNA sequence data from at least two different V_(H) gene clones,from the hybridoma cell line expressing the murine BAT antibody, matchedperfectly were the gene sequences accepted as correct.

[0094] Three separate PCR-products, each from a different total RNApreparation and subsequent first strand cDNA synthesis reaction, werePCR-cloned and completely DNA sequenced on both strands. Although alltwelve heavy chain primers were tested (Table 1), only the MHV9 primer(in conjunction with MHCG3—designed to anneal to the CH₁ domain of themouse γ3 heavy chain gene) was PCR-amplified an approximately 460 bpproduct which was then TA-cloned into the pCRII™ cloning vector (datanot shown).

[0095] DNA sequence analysis of several individual clones from each ofthe three PCR-products (each from different 1^(st) strand synthesisreactions and subsequent PCR-reactions) resulted in the determination ofthe BAT antibody heavy chain variable region sequence as described inFIG. 1. This sequence was confirmed on both DNA strands for all threePCR-clones studied.

EXAMPLE 2 Cloning and Sequencing of the Kappa Light Chain VariableRegion of the BAT Antibody

[0096] The single stranded cDNA template, produced via 1^(st) strandsynthesis, was PCR-amplified using a series of kappa light chaindegenerate primers (Table 2 below). However, this resulted in theamplification of a number of PCR-products from more than one degenerateprimer, suggesting that more than one variable region gene was beingtranscribed, at least, by the BAT hybridoma cell line.

[0097] First, a PCR-product was seen when the MKV2 primer (which, likeall of the MKV series of primers, anneals to the 5′ end of the DNAsequence of the kappa light chain signal peptide) and MKC (which isdesigned to anneal to the 5′ end of the mouse kappa constant regiongene) were used together. Previous in-house experience had shown us thatthe MKV2 primer would PCT-amplify an aberrant mRNA transcript. Thisaberrant pseudogene was present in all standard fusion partners derivedfrom the original MOPC-21 plasmacytoma cell line and was known asMOPC-21n Deyev, S. M., et al., Genetica, 85:45, 1991. NO-0 was a cellline which was derived from MOPC-21 line, and it was this line which wasused as the fusion partner to produce the BAT hybridoma. Consequently,it was not surprising that a PCR-product was seen when using the MKV2primer. This product was analyzed and shown to be the non-functionalpseudogene (data not shown).

[0098] Unusually, another pseudogene, previously identified as beingsecreted by the related cell line NS-1 Hamlyn, P. H., et al., Nucl. AcisRes., 9:4485, 1981 and normally PCR-cloned when using the MKV7 primer inconjunction with MKC primer, was not seen in any of the PCR-products sofar analyzed. Since the NS-1 and NS-0 cell lines were very closelyrelated, this was a little surprising. However, it also highlighted theconfusing nature of kappa light chain transcription that was present inthe BAT hybridoma cell line.

[0099] Another PCR-clone, which ultimately turned out to be the V_(K)gene of the BAT antibody, was also successfully PCR-amplified from theBAT hybridoma cell line with the primers MKV5 and MKC. Followingtransformation of the approximately 450 bp product into INVαF′ competentcells, putative positive transformants were identified using thePCR-screening assay and then DNA sequenced.

[0100] From sequence analysis of two individual clones of the MKV5product (each from different ₁ ^(st) strand synthesis reactions andsubsequent PCR-reactions) the DNA sequence of the BAT antibody kappalight chain variable region gene was determined (FIG. 2). This sequencewas again confirmed on both DNA strands for each clone.

EXAMPLE 3 Sequence Analysis of the Mouse BAT Antibody Variable Regions

[0101] The amino acid sequence of the BAT V_(κ) and V_(H) regions werecompared to the consensus sequences of murine variable region subgroupsthat were defined in the Kabat (Supra) database From this analysis theBAT V_(H) region was found to most closely match the consensus sequenceof mouse kappa subgroup VI. Similar comparisons of the BAT V_(H) regionto the Kabat databasefound that it exhibited the closest match to theconsensus sequence of mouse heavy chain subgroup “miscellaneous”.

[0102] A comparison of the above BAT antibody variable region sequencesto a database of murine germlines, found that the closest germline geneto the BAT V_(H) gene was VMS/VGK4 (FIG. 3), whilst the closest germlinegene to the BAT V_(κ) gene was H4 (FIG. 4). As can be seen in FIG. 3,those mismatches that did occur between the BAT V_(H) gene and itsclosest germline gene were, unsurprisingly, predominantly located in theCDR2 and CDR3. There were only three framework changes, and all thesewere located in FR3. With respect to the BAT V_(κ) gene (FIG. 4), it wasagain not all together surprising that the majority of mismatches werepositioned in the CDRs. The four differences that were located in theFRs were all highly conservative changes, except for the cysteine atposition 72 (Kabat numbering) in FR3. Its location immediately adjacentto an important canonical residue (position 71) suggested that thecysteine may have been playing a key role in antigen binding. However,only through modeling the Fv domain could such a supposition beclarified.

[0103] Nevertheless, these analyses confirmed that both the V_(H)regions and the V_(κ) regions of the mouse BAT variable regions appearedto be typical of mouse variable regions. TABLE 1 PCR-primers used in thecloning of the BAT heavy chain variable region gene Name Sequence(5′→3′) MHV5^(a)  (30 mer; SEQ ID NO:12) ATGGACTCCAGGCTCAATTTAGTTTTCCTTMHV9^(a)  (30 mer; SEQ ID NO:13) ATGGATTGGGTGTGGACCTTGCTATTCCTG    C           A MHCG3^(b) (21 mer; SEQ ID NO:14) CAAGGGATAGACAGATGGGGC

[0104] TABLE 2 PCR-primers used in the cloning of the BAT kappa lightchain variable region gene Name Sequence (5′→3′) MKV2^(a)  (30 mer; SEQID NO:15) ATGGAGACAGACACACTCCTGCTATGGGTG       T              T MKV5^(a) (30 mer; SEQ ID NO:16) ATGGATTTTCAGGTGCAGATTATCAGCTTC        A            T MKV6^(a)  (30 mer; SEQ ID NO:17)ATGAGGTGCCCTGTTCAGTTCCTGGGG    T TT  C G  C T   A MKV11^(a) (30 mer; SEQID NO:18) ATGGAAGCCCCAGCTCAGCTTCTCTTCC MKC^(b)   (20 mer; SEQ ID NO:19)ACTGGATGGTGGGAAGATGG

[0105] TABLE 3 Primers for PCR screening transformed colonies NameSequence (5′-43 3′) pCRII Forward Primer (18 mer; SEQ ID:20)CTAGATGCATGCTCGAGC pCRII Reverse Primer (21 mer; SEQ ID:21)TACCGAGCTCGGATCCACTAG

II. CONSTRUCTION AND EXPRESSION OF THE CHIMERIC ANTIBODY OF THEINVENTION

[0106] (A) Abbreviations

[0107] The following non-SI unit and other abbreviations were used:

[0108] Polymerase chain reaction (PCR); deoxyribonucleic acid (DNA);copy DNA (cDNA); kappa light chain variable region (V_(κ)); heavy chainvariable region (V_(H)); minute (min); Tris-borate buffer (TBE);phosphate buffered saline (PBS); room temperature (RT), bovine serumalbumin (BSA); hydrochloric acid (HCl); horseradish peroxidase (HRP);low fat milk LFM); hour (hr); percent (%); O-phenylenediaminedihydrochloride (OPD); multiple cloning site (MCS).

[0109] (B) Materials

[0110] Media components and all other tissue culture materials wereobtained from Life Technologies (UK). The constituents for thePCR-reactions, including AmpliTaq® DNA polymerase, were purchased fromPerkin Elmer (USA). However, the TA Cloning® kit and INVαF′ competentcells were obtained from Invitrogen (USA). DH5α competent cells andagarose (UltraPure™) were obtained from Life Technologies (UK). TheThermo Sequenase™ pre-mixed cycle sequencing kit and the Vistra 725 DNAsequencing machine were both purchased from Amersham (UK). The Big Dye™Terminator Cycle Sequencing Ready Reaction Kit used with the ABI Prism310 Genetic Analyzer were purchased from PE Applied Biosystems (UK). Allother molecular biological products described were obtained either fromNew England biolabs (USA) or Promega (USA). Nunc-Immuno Plate MaxiSorp™immunoplates were purchased from Life Technoloiges (UK) while the Comingeasy wash ELISA plates were obtained from Coming Laboratory SciencesCompany (UK). The goat anti-human IgG (Fc_(γ) fragment specific)antibody, the goat anti-human kappa light chain/HRP conjugate and theAffinPure goat anti-human IgG (Fc_(γ) fragment specific)/HRP conjugatewere obtained from Jackson ImmunoResearch Laboratories Inc. (USA).K-Blue TMB substrate and Red Stop solution were purchased from NeogenInc. (USA). All other products for the ELISA were obtained from Sigma(UK). Microplate Manager® data analysis software package was purchasedfrom Bio-Rad (UK). The micro-volume stirred ultrafiltration cell andPM30 filter membrane were obtained from Amicon PLC (UK), while theImmunopure® (G) IgG purification kit was purchased from Pierce PLC (UK).

[0111] (C) Experimental Techniques

[0112] C1 Construction of chimeric γ1/_(κ) BAT antibody

[0113] The previously isolated mouse kappa light chain variable region(V_(κ)) gene (FIG. 1) and heavy chain variable region (V_(H)) gene (FIG.2) were modified at the 5′- and 3′-ends, using specifically designedPCR-primers (Table 1), to enable expression of the BAT variable regiongenes in mammalian cells as part of a chimeric mouse-human antibody. Toachieve this separation PCR-reactions were prepared for each variableregion gene in a specific PCR-room using specific protocols designed tominimize the possibility of cross-contamination. The plasmidsBATV_(H)-pCR2. 1 and BATV_(κ)-pCR2. 1 were used as templates andAmpliTaq® DNA polymerase was used t amplify these templates. PrimersB8814 and B8815 (Table 4) were used to PCR-modify the BAT V_(H) genewhile primers C0224 and C0225 (Table 4) were used to PCR-mutate the BATV_(κ) gene.

[0114] The PCR-reaction tubes were cycled (after an initial melt at 94°C. for 3 min) at 94° C. for 50 s, 72° C. for 1 min 30 s over 30 cycles.At the completion of the last cycle a final extension step at 72° C. for10 min was carried out before the reactions were cooled on ice. 5 μlaliquots from each PCR-reaction were then run on a 1.2% agarose/TBE (pH8.8) gel to determine which had produced a PCR-product of the correctsize.

[0115] 1-2 μl aliquots of those PCR-products of the correct size weredirectly cloned into the pCR2.1™ vector, provided by the TA Cloning®kit, and transformed into INVαF′ competent cells as described in themanufacturers instructions. Colonies containing the plasmid, with acorrectly sized insert, were identified by PCR-screening the coloniesusing the 1212 and 1233 oligonucleotide primers (Table 5) according tothe method of Güissow and Clackson (Supra) Those putative positiveclones identified were double-stranded plasmid DNA sequenced using boththe Vistra DNA sequencing machine and ABI Prism 310 Genetic Analyzer.The Thermo Sequenase™ pre-mixed cycle sequencing kit and the Big Dye™Terminator Cycle Sequencing Ready Reaction Kit were used as described inthe manufacturers instructions with the primers 1212 and 1233 (Table 5).

[0116] Those clones containing the correctly adapted BAT V_(κ) and V_(H)genes (FIGS. 5 and 6, respectively) were subcloned, as a HindIII-BamHifragments, into the expression vectors pKN110 (FIG. 7) and pG1D110 (FIG.8), respectively, to express chimeric light and heavy chains inmammalian cells. The ligated expression vectors (i.e. pKN 110-BATV_(κ)and pG1D110-BATV_(H)) were then transformed into DH5_(α) competentcells. Positive clones, containing the correctly constructed expressionvectors, were finally identified by restriction digest analysis.

[0117] C2 Co-transfection of chimeric γ1/_(κ) BAT antibody vector DNAinto COS cells

[0118] The method of Kettleborough et al. was followed to transfect themammalian expression vectors into COS cells. Briefly, the DNA (10 μgeach of the kappa light chain expression vector pKN110-BATV_(κ) andheavy chain expression vector pG1D110-BATV_(H)) was added to a 0.70 mlaliquot of 1×10⁷ cells/ml in PBS and pulsed at 1900 V, 25 μF capacitanceusing a Bio-Rad Cene Pulser apparatus. Following a 10 min recovery at RTthe electroporated cells were added to 8 ml of DMEM containing 5% FCSand incubated for 72 hr in 5% CO₂ at 37° C. After 72 hr incubation, themedium was collected, spun to remove cell debris and analyzed by ELISAfor chimeric BAT antibody production.

[0119] C3 Quantification of chimeric γ1/_(κ) antibody via ELISA

[0120] Each well of a 96-well Nunc-Immuno Plate MaxiSorp™ immunoplate asfirst coated with 100 μl aliquots of 0.4 ng/μl goat anti-human IgG(Fc_(γ) fragment specific) antibody, diluted in PBS and incubatedovernight at 4° C. and removed prior to use. 100 μl/well aliquots f theexperimental samples (i.e. harvested COS cell supernatants—spun toremove cell debris) and 1:2 sample dilutions, diluted in sample-enzymeconjugate buffer (0.1 M Tris-HCl (pH 7.0), 0.1 M NaCl, 0.02% (v/v)TWEEN-20 and 0.2% (w/v) BSA), were then dispensed onto the immunoplate.In addition, a purified human γ1/_(κ) antibody (1000 ng/μl), which wasused as a standard and serially diluted 1:2, and also loaded onto theimmunoplate. The immunoplate was incubated at 37° C. for 1 hr beforebeing washed with 200 μl/well of wash buffer (PBS/0.1% (v/v) TWEEN-20)three times. 100 μl of goat anti-human kappa light chain/horseradishperoxidase conjugate, diluted 5000-fold in sample-enzyme conjugatebuffer, was added to each well, following which the immunoplate wasincubated at 37° C. for 1 hr before it was washed as before. 150 μlaliquots of K-Blue substrate were then added to each well, followingwhich the immunoplate was incubated for 10 min at RT in the dark. Thereaction was finally halted by dispensing 50 μl of Red Stop into eachwell. The optical density at 655 nm was then determined using a Bio-Rad3550 microplate reader in conjunction with the Microplate Manager®software package.

[0121] C4 Purification of the Chimeric BAT Antibody

[0122] The chimeric BAT γ1/_(κ) antibody was purified from COS cellsupernatants in two stages. First, a micro-volume stirredultrafiltration cell with a PM30 filter membrane was used, according tothe manufacturers instructions, to reduce the volume of the raw,non-purified supernatant. Then an Immunopure® (G) IgG purification kitwas used to affinity purify the chimeric BAT antibody from theconcentrated supernatant, also according to the manufacturersinstructions.

[0123] C5 Daudi cell ELISA

[0124] The cell ELISA assay was carried out using the Daudi cellcultured from an original stock also by Dr. Hardy (Felsenstein MedicalResearch Center, Rabin Medical Center, Beilinson Campus, PetachTikva,49100, Israel). Minor modifications were made to the assaydepending upon whether the mouse or the mouse-human chimeric BATantibody was being analyzed. When assaying the binding affinity of themouse BAT antibody a goat anti-mouse IgG (Fab specific)/HRP conjugate(diluted 1:15000) was used as the secondary antibody. Conversely, whenmeasuring the affinity of the chimeric BAT antibody AffiniPure goatanti-human IgG (Fc_(γ) fragment specific)/HRP conjugate (diluted1000-fold) was used.

[0125] The Daudi cells (2 days after being passaged) were first platedat 10⁵ cells/well in a 96 well Coming easy wash ELISA plate and thenincubated overnight at 37° C. in a dry incubator. The next day, 200 μlof rehydration buffer (PBS containing 10% FCS and 0.05% azide) was addedto each well which was then left for a minimum of 1 hr. The rehydrationbuffer was then decanted off before 50 μl aliquots of various 1:2 serialdilutions of the purified BAT antibody was added to the wells of theplate. The plate was again incubated overnight (at 4° C.), washed twicewith 200 μl/well of PBS containing 5% LFM and allowed to dry. 50 μl/wellof the HRP conjugated secondary antibody was then added before a seriesof six different washes (i.e. one wash with PBS containing 5% LFM, threewashed with the same buffer supplemented with 0.05% TWEEN-20, followedby a further two washes with the PBS/LFM buffer) were carried out. 200μl/well of 0.4 mg/ml OPD substrate in 0.05 M citrate buffer (pH 5.0) and60 mg/ml hydrogen peroxide was then added before the ELISA plate wasincubated in the dark and at RT until the color had developed (usuallyabout 30 min). Finally, the reaction was stopped by the addition of 50μl/well of 2.5 M sulfuric acid and the optical density at 490 nm wasthen measured using a Bio-Rad 3550 microplate reader in conjunction withthe Microplate Manager® software package.

[0126] Results

EXAMPLE 4 Construction of the Chimeric γ1/_(κ) BAT antibody

[0127] As with all projects, a strict PCR-cloning and sequencingprotocol was followed. This was done to minimize the possibility ofintroducing errors into the wild-type sequences of the mouse variableregion genes during the PCR-modification step. Using the primers C0224and C0225 (Table 1) the mouse BAT V_(κ) gene (FIG. 2) was modified viaPCR to produce a 418 bp band (data not shown). This PCR-product wasligated into the pCR2.1 plasmid and transformed into INVαF′ competentcells. Similarly, the mouse BAT V_(H) gene (FIG. 1 was PCR-mutated usingprimers B8814 and B8815 (Table 1) to produce a 436 bp band (data notshown). This PCR-product was also ligated into the pCR2.1 plasmid andtransformed into INVαF′ competent cells.

[0128] Putative positive transformants were then detected using thePCR-screening assay (data not shown) before finally being ds-DNAsequenced on the ABI Prism 310 Genetic Analyzer. FIGS. 3 and 4 show theresults of this DNA sequence analysis of the chimeric BAT V_(κ) gene andBAT V_(H) gene, respectively. The analysis was carried out both toconfirm their successful mutagenesis and also show the presence of anyPCR-errors that may have been introduced into the genes. Only onePCR-reaction was actually carried out for each variable region gene andonly two clones from each of these PCR-reactions were eventually DNAsequences to completed.

[0129] Nevertheless, this proved sufficient to isolate at least oneclone for each modified variable region gene which contained the correctmodified DNA sequence.

[0130] The mutated V_(H) and V_(κ) genes were then subcloned into theappropriate expression vectors, as hindIII/BamHI fragments, to createpKN110-BATV_(κ) (7.88kb) and pG1D110-BATV_(H) (7.55 kb), respectively.The fidelity of the expression vectors constructed was then confirmedvia restriction enzyme analysis (data not shown). Once co-transfectedinto COS cells, these vectors wold allow the transient expression of aγ1/_(κ) version of the chimeric BAT antibody.

[0131] In addition, as an extra component to the BAT antibodyhumanization project, the BAT V_(H) gene was also subcloned, as aHindIII/BamHI fragment, into both the pG3D110 and the pG4D1100 heavychain expression vectors. These vectors were identical to pG1D110, savefor the replacement of the cDNA copy γ1 human constant region genes witheither a cDNA copy of the ³γ constant region genes (in the case ofpG3D110) or the cDNA of the γ3 constant region genes (in the case ofpG3D110) or the cDNA of the γ4 constant region genes (in the case ofpG3D110). The construction of these vectors (i.e. pG3D110-BATV_(κ), ofboth γ3/_(κ) and γ4_(κ) versions of the chimeric BAT antibody in COScells.

EXAMPLE 5 Transient Expression of the Chimeric γ1/_(κ) BAT Antibody

[0132] The two vectors pKN110-BATV_(κ) and pG1D110-BATV_(H) wereco-transfected into COS cells in a series of repeated transientexpression experiments. After being expressed for 72 hr the mouse-humanγ1/_(κ) chimeric BAT antibody was detected in the supernatant of the COScell co-transfections via the γ1/_(κ) ELISA. From these assays the meanconcentration of γ1/_(κ) chimeric BAT antibody detected in the media wascalculated to be 509±272 ng/ml.

[0133] Interestingly, the γ3/_(κ) and γ4/_(κ) versions of the chimericBAT antibody appeared to produce significantly greater quantities ofantibody following their expression COS cells. Specifically, whenpG3D110-BATV_(H) and pKN110BATV_(κ) were co-transfected into COS cells,initial analysis of the supernatant (using the ELISA method described inSection 4.3 and human IgG3/kappa antibody as a standard) measured theexpression levels of the chimeric γ3/_(κ) BAT antibody to be 6.7 μg/ml.Moreover, when pG4D110-BATV_(H) pKN110-BATV_(κ) were expressed in COScells, the same ELISA (using human IgG4/kappa antibody as a standard)measured the expression levels of the chimeric γ4/_(κ) BAT antibody tobe 8.2 μg/ml.

EXAMPLE 6 Purification of the Chimeric γ1/_(κ) BAT antibody

[0134] Harvesting approximately 8 ml per co-transfection, a series oftransfections were carried out until 200 ml of COS supernatant had beencollected. The volume of this supernatant was then reduced to 15 ml bypassing the supernatant through a micro-volume stirred ultrafiltrationcell with a PM30 filter membrane—which had a molecular weight cut-off of30 kDa.

[0135] The Immunopure® (G) IgG purification kit essentially comprised ofa 2 ml column of immobilized Protein G column. The antibody was elutedfrom the column with 6 ml of elution buffer, the eluate of which wascollected in 1 ml fractions. The concentration of chimeric γ1/_(κ) BATantibody in each fraction was then assayed using the ELISA methoddescribed in Section C3. This analysis found that the chimeric antibodywas present in Fraction 3 (42.05 μg/ml) and Fraction 4 (20.05 μg/ml),which correspond to a total recovery of 62.1 μg of chimeric γ1/_(κ) BATantibody. This was stored at −20° C., until its subsequent transfer toCuretech for further analysis.

EXAMPLE 7 Analysis of Daudi Cell Binding by the Chimeric γ1/_(κ) Batantibody

[0136] Using the Daudi cell ELISA it was clearly shown that the purifiedchimeric γ1/_(κ) BAT antibody bound to Daudi cells. FIG. 9 shows atypical example of one experiment. However, what was less conclusive wasthe binding of similar concentrations of mouse BAT antibody, in the sameELISA, which appeared to be lower than the chimeric antibody.Nevertheless, since the conjugated secondary antibody used to detectantibody binding to the Daudi cells was different for each antibodyconstruct, no direct comparison of the binding of the two versions canlegitimately be made. TABLE 4 Primers used to PCR-modify the mouse BATantibody kappa light chain and heavy chain variable region genes toallow their expression as part of a chimeric γ1/₇₈ BAT antibody inmammalian cells Name Sequence (5′→3′) C0225 (42 mer; SEQ ID:22) C C C AA G C T T G C C G C C A C C A T G G A T T T T C A G G T G C A G A T T AT C C0224 (39 mer; SEQ ID NO:23) C G C G G A T C C A C T C A C G T T T TA T T T C C A A C T T T G T C C C C G B8815 (40 mer; SEQ ID NO:24) G G AT C C A C T C A C C T G A G G A G A C G G T G A C T G A G G T T C C T TG B8814 (42 mer; SEQ ID NO:25) A A G C T T G C C G C C A C C A T G G C TT G G G T G T G G A C C T T G C T A T T C

[0137] TABLE 5 Primers used to PCR screen the transformed colonies andDNA sequence the PCR-modified variable region genes of the BAT antibodyName Sequence (5′→3′) Huγ1 (17 mer; SEQ ID NO:26) T T G G A G G A G G GT G C C A G HCMVi.3s (28 mer; SEQ ID NO:27) G T C A C C G T C C T T G AC A C G C G T C T C G G G A FOR (18 me; SEQ ID NO:28) T G T A A A A C GA C G G C C A G T REV (18 mer; SEQ ID NO:29) G A A A C A G C T A T G A CC A T G B6990 (27 mer; SEQ ID NO:30) C A G C A T A T G T T G A C T C T CC A C T G T C G G B6991 (27 mer; SEQ ID NO:31) G T C A A C A T A T G C TG A A G A G T T C A A G G G B8809 (18 mer; SEQ ID NO:32) T G C C A G G TC A A G T G T A A G B8810 (18 mer; SEQ ID NO:33) A A G C C A G G T T G GA T G T C C

IV AMINO ACID SEQUENCES OF 3 PEPTIDES TAKEN FROM THE DAUDI B-CELLLYMPHOBLASTOID CELL LINE ANTIGEN TO WHICH THE MABS OF THE INVENTION BIND

[0138] Three peptides comprised in the antigenic epitope of the Daudi Blymphoblastoid cells to which the mAbs of the invention bind weresequenced. Their sequence depicted in FIGS. 10, 11 and 12.

[0139] Searches performed against the non-redundant gene bank databaseand the EST Division yielded no hits when the three peptides were ran asqueries using the TBLASTN algorithm (Version 2) with an EXPECT value of10 and the matrix BLOSUM 62.

[0140] However, since the peptides are small peptides, they weresubmitted again at a higher EXPECT value to make the search lessstringent. The filter was also unmasked for low complexity which caneliminate potentially confounding matches (e.g. hits againstproline-rich regions or proly-A tails) from the blast reports, leavingregions whose blast statistics reflect the specificity of their pairwisealignment. The three peptides of the invention did not yield any hitwith the gene bank and the EST division database even at a very lowstringency.

[0141] Thus, in accordance with the above results, the three abovepeptides seem to be novel peptides.

IV DIAGNOSIS OF MALIGNANT DISEASES IN PATIENTS USING THE MAB OF THEINVENTION

[0142] Peripheral blood lymphocytes from tested individuals weredouble-labeled using the anti-CD3 antibody and one of the mAbs of theinvention. The percent of CD3⁺ cells which bind the mAbs of theinvention were determined. In accordance with the invention, it has beenshown that the number of the CD3^(+mAb) ⁺ cells in individuals having amalignant disease differs from the percent of these cells in bloodsamples obtained from healthy individuals. The fact that there exists asignificant difference of the percent of the CD3⁺ cells in theindividuals having a malignant disease and whether the difference isabove or below the percent of CD3⁺mAb⁺ cells obtained from healthyindividuals enables to determine at high probability whether theindividual has a malignant disease as well as the specific kind ofmalignant disease which the tested individual may have.

[0143] Typically, human peripheral blood lymphocytes were obtained from20 ml blood of either a healthy individual or from cancer patients byFicoll Hypaque density centrifugation. The cells were washed andsuspended in PBS containing 0.5% BSA and 0.05% as acid. The samplescontaining 0.5×10⁶ cells were used for FACS analysis. First, the cellswere incubated with a saturated amount of the mAb of the invention for45 mins. at 0° C. followed by their incubation with an anti-mouse mAbconjugated to FITC for 30 mins. on ice. After two washes andcentrifugation at 1200 rpm cells were incubated with an anti-human CD3conjugated to PE antibodies for 30 mins. on ice. Following thisincubation, the cells were washed twice and the sample is analyzed by aFACS scan (Bectan Dickinson). The results are shown in FIGS. 13 to 17.

[0144] As can be seen in FIG. 13, as well as in FIG. 17, the percent ofCD3⁺ BAT⁺cells (as compared to total CD3⁺ cells) in blood samplesobtained from healthy individuals is in the range of about 25%. As seenin FIG. 14, the percent of the CD3⁺ BAT⁺ cells in blood samples obtainedfrom patients having colon carcinoma is substantively lower, as comparedto healthy individuals, in the range of about 7%. Similarly, the percentof CD3⁺ BAT⁺ cells in blood samples obtained from patients having breastcarcinoma was in the range of about 10% (FIG. 15). These results clearlyindicate that colon and breast carcinoma can be identified by the factthat the percent of CD3⁺ BAT⁺ cells is lower as compared to healthyindividuals.

[0145] The percent of CD3⁺ BAT⁺ cells in blood samples obtained fromprostate carcinoma patients is significantly higher than thepercentagein blood samples of healthy individuals as seen in FIG. 16 andis in the range of about 50%. These results clearly indicate thatprostate carcinoma can be identified by the fact that the percent ofCD3⁺ BAT⁺ cells is higher a compared to healthy individuals. As seen inFIG. 18, the amount of the antigen to, which the mAb of the inventionbind found on T-cells, obtained from prostate carcinoma patients is veryhigh while the antigen is undetectable in T-cells obtained from patientsof breast carcinoma.

[0146] The above results show that the mAbs of the invention may be usedin order to identify an individual suffering from a certain kind ofmalignant disease. Thus, if a blood sample is obtained from a testedindividual and the extent of binding of the mAbs of the invention toCD3⁺ cells in the sample is significantly high (in the range of about50%), there is a very high probability that the tested individual issuffering from prostate cancer. Against this, if the percent of the CD3⁺cells in the sample is significantly low as compared to healthyindividuals (in the range of about 7% or 10%), there is a highprobability that the tested individual is suffering from breast or coloncarcinoma. Obviously, if the tested individual is a male individual,there is a high probability of his suffering from colon carcinoma.

[0147] The above examples are not to be construed as limiting andadditional correlations between the percent of CD3⁺ cells which bind themAbs of the invention and other malignant diseases are also within thescope of the invention.

1 33 1 462 DNA Humanus 1 tactagtcga catggcttgg gtgtggacct tgctattcctgatggcagct gcccaaagta 60 tccaagcaca gatccagttg gtgcagtctg gacctgagttgaagaagcct ggagagacag 120 tcaagatctc ctgcaaggct tctggatata ctttcacaaactatggaatg aactgggtga 180 agcaggctcc aggaaagggt ttaaagtgga tgggctggataaacaccgac agtggagagt 240 caacatatgc tgaagagttc aagggacggt ttgccttctctttggaaacc tctgccaaca 300 ctgcctattt gcagatcaac aacctcaaca atgaggacacgcctacatat ttctgtgtga 360 gagtcggcta cgatgctttg gactactggg gtcaaggaacctcagtcacc gtctcctcaa 420 ctacaacaac agccccatct gtctatccct tcccgggttc ca462 2 136 PRT Humanus 2 Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met AlaAla Ala Gln Ser 1 5 10 15 Ile Gln Ala Gln Ile Gln Leu Val Gln Ser GlyPro Glu Leu Lys Lys 20 25 30 Pro Gly Glu Thr Val Lys Ile Ser Cys Lys AlaSer Gly Tyr Thr Phe 35 40 45 Thr Asn Tyr Gly Met Asn Trp Val Lys Gln AlaPro Gly Lys Gly Leu 50 55 60 Lys Trp Met Gly Trp Ile Asn Thr Asp Ser GlyGlu Ser Thr Tyr Ala 65 70 75 80 Glu Glu Phe Lys Gly Arg Phe Ala Phe SerLeu Glu Thr Ser Ala Asn 85 90 95 Thr Ala Tyr Leu Gln Ile Asn Asn Leu AsnAsn Glu Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Val Arg Val Gly Tyr AspAla Leu Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Ser Val Thr Val Ser Ser130 135 3 443 DNA Humanus 3 actagtcgac atggatttac aggtgcagat tatcagcttcctgctaatca gtgcctcagt 60 cataatgtcc agaggacaaa ttgttctcac ccagtctccagcaatcatgt ctgcatctcc 120 aggggagaag gtcaccataa cctgcagtgc caggtcaagtgtaagttaca tgcactggtt 180 ccagcagaag ccaggcactt ctcccaaact ctggatttataggacatcca acctggcttc 240 tggagtccct gctcgcttca gtggcagtgg atctgggacctcttactgtc tcacaatcag 300 ccgaatggag gctgaagatg ctgccactta ttactgccagcaaaggagta gtttcccact 360 cacgttcggc tcggggacaa agttggaaat aaaacgggctgatgctgcac caactgtatc 420 catcttccca ccatccaaga tct 443 4 128 PRTHumanus 4 Met Asp Leu Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser AlaSer 1 5 10 15 Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser ProAla Ile 20 25 30 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys SerAla Arg 35 40 45 Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro GlyThr Ser 50 55 60 Pro Lys Leu Trp Ile Tyr Arg Thr Ser Asn Leu Ala Ser GlyVal Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr CysLeu Thr Ile 85 90 95 Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr CysGln Gln Arg 100 105 110 Ser Ser Phe Pro Leu Thr Phe Gly Ser Gly Thr LysLeu Glu Ile Lys 115 120 125 5 412 DNA Humanus 5 aagcttgccg ccaccatggatttacaggtg cagattatca gcttcctgct aatcagtgcc 60 tcagtcataa tgtccagaggacaaattgtt ctcacccagt ctccagcaat catgtctgca 120 tctccagggg agaaggtcaccataacctgc agtgccaggt caagtgtaag ttacatgcac 180 tggttccagc agaagccaggcacttctccc aaactctgga tttataggac atccaacctg 240 gcttctggag tccctgctcgcttcagtggc agtggatctg ggacctctta ctgtctcaca 300 atcagccgaa tggaggctgaagatgctgcc acttattact gccagcaaag gagtagtttc 360 ccactcacgt tcggctcggggacaaagttg gaaataaaac gtgagtggat cc 412 6 128 PRT Humanus 6 Met Asp LeuGln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val IleMet Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30 Met SerAla Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Arg 35 40 45 Ser SerVal Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser 50 55 60 Pro LysLeu Trp Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Cys Leu Thr Ile 85 90 95 SerArg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg 100 105 110Ser Ser Phe Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 115 120125 7 436 DNA Humanus 7 aagcttgccg ccaccatggc ttgggtgtgg accttgctattcctgatggc agctgcccaa 60 agtatccaag cacagatcca gttggtgcag tctggacctgagttgaagaa gcctggagag 120 acagtcaaga tctcctgcaa ggcttctgga tatactttcacaaactatgg aatgaactgg 180 gtgaagcagg ctccaggaaa gggtttaaag tggatgggctggataaacac cgacagtgga 240 gagtcaacat atgctgaaga gttcaaggga cggtttgccttctctttgga aacctctgcc 300 aacactgcct atttgcagat caacaacctc aacaatgaggacacggctac atatttctgt 360 gtgagagtcg gctacgatgc tttggactac tggggtcaaggaacctcagt caccgtctcc 420 tcaggtgagt ggatcc 436 8 135 PRT Humanus 8 MetAla Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser 1 5 10 15Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys 20 25 30Pro Gly Glu Thr Val Glu Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu 50 55 60Lys Trp Met Gly Trp Ile Asn Thr Asp Ser Gly Glu Ser Thr Tyr Ala 65 70 7580 Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn 85 9095 Thr Ala Tyr Leu Gln Ile Asn Asn Leu Asn Asn Glu Asp Thr Ala Thr 100105 110 Tyr Phe Cys Val Arg Val Gly Tyr Asp Ala Leu Asp Tyr Trp Gly Gln115 120 125 Gly Thr Ser Val Thr Val Ser 130 135 9 9 PRT Humanus 9 ThrIle Asn Glu Glu Glu Glu Lys Cys 1 5 10 14 PRT Humanus 10 Asn Ser Gly ProSer Met Arg Lys Lys Asn Val Ser Ile Gly 1 5 10 11 5 PRT Humanus 11 IlePro Asp His Gln 1 5 12 30 DNA artificial sequence PCR-primers used inthe cloning of the BAT heavy chain variable region gene 12 atggactccaggctcaattt agttttcctt 30 13 30 DNA artificial sequence PCR-primers usedin the cloning of the BAT heavy chain variable region gene 13 atggattgggtgtggacctt gctattcctg 30 14 21 DNA Artificial Sequence PCR-primers usedin the cloning of the BAT heavy chain variable region gene 14 caagggatagacagatgggg c 21 15 30 DNA Artificial Sequence PCR-primers used in thecloning of the BAT kappa light chain variable region gene 15 atggagacagacacactcct gctatgggtg 30 16 30 DNA Artificial Sequence PCR-primers usedin the cloning of the BAT kappa light chain variable region gene 16atggattttc aggtgcagat tatcagcttc 30 17 27 DNA Artificial SequencePCR-primers used in the cloning of the BAT kappa light chain variableregion gene 17 atgaggtgcc ctgttcagtt cctgggg 27 18 28 DNA artificialsequence PCR-primers used in the cloning of the BAT kappa light chainvariable region gene 18 atggaagccc cagctcagct tctcttcc 28 19 18 DNAArtificial Sequence PCR-primers used in the cloning of the BAT kappalight chain variable region gene 19 ctagatgcat gctcgagc 18 20 20 DNAArtificial Sequence pCRII Forward Primer 20 actggatggt gggaagatgg 20 2121 DNA Artificial Sequence pCRII Reverse Primer 21 taccgagctc ggatccactag 21 22 42 DNA artificial sequence C0225 primer 22 cccaagcttg ccgccaccatggattttcag gtgcagatta tc 42 23 39 DNA artificial sequence C0224 Primer23 cgcggatcca ctcacgtttt atttccaact ttgtccccg 39 24 40 DNA artificialsequence B8815 Primer 24 ggatccactc acctgaggag acggtgactg aggttccttg 4025 42 DNA artificial sequence B8814 Primer 25 aagcttgccg ccaccatggcttgggtgtgg accttgctat tc 42 26 17 DNA artificial sequence Huy1 Primer 26ttggaggagg gtgccag 17 27 28 DNA artificial sequence HCMVi.3s primer 27gtcaccgtcc ttgacacgcg tctcggga 28 28 18 DNA artificial sequence FORprimer 28 tgtaaaacga cggccagt 18 29 18 DNA artificial sequence REVprimer 29 gaaacagcta tgaccatg 18 30 27 DNA artificial sequence B6990primer 30 cagcatatgt tgactctcca ctgtcgg 27 31 27 DNA artificial sequenceB6991 primer 31 gtcaacatat gctgaagagt tcaaggg 27 32 18 DNA artificialsequence B8809 primer 32 tgccaggtca agtgtaag 18 33 18 DNA artificialsequence B8810 primer 33 aagccaggtt ggatgtcc 18

1. A monoclonal antibody comprising a heavy chain variable region havingat least 70% identity to the amino acid sequence of FIG. 1; a lightchain variable region having at least 70% identity to the amino acidsequence of FIG. 2; or both said heavy chain variable region and saidlight chain variable region.
 2. The monoclonal antibody according toclaim 1, wherein said heavy chain variable region comprises of the aminoacid sequence of FIG. 1; said light chain variable region comprises ofthe amino acid sequence of FIG. 2; or both said heavy chain variableregion and said light chain variable region comprising FIG. 1 and FIG. 2correspondingly.
 3. A monoclonal antibody, wherein said antibody bindsto an antigen to which any one of the monoclonal antibodies of claim 1specifically bind.
 4. The antibody according to claim 1, wherein theantibody is a chimeric human-mouse antibody.
 5. A nucleic acid moleculeencoding the amino acid sequence of the monoclonal antibody of claim 1.6. An expression vector having the nucleic acid sequence of claim
 5. 7.An expression vector according to claim 6, comprising of plasmid pKN110or plasmid pG1D110.
 8. A cell transfected with the expression vector ofclaim
 6. 9. A hybridoma cell line producing at least one of themonoclonal antibodies of claim
 1. 10. A peptide having an amino acidsequence at least 85% identical to the amino acid sequence of thepeptides in FIG. 10, FIG. 11, or FIG.
 12. 11. The peptide of claim 10,wherein the peptide comprises of the amino acid sequence of the peptidein FIG. 10, FIG. 11, or FIG.
 12. 12. A protein or peptide comprising oneor more of the peptides of claim
 10. 13. A peptide analog of any of thepeptides of claim 10, wherein said peptide analog has substantially thesame level of binding to a monoclonal antibody comprising a heavy chainvariable region having at least 70% identity to the amino acid sequenceof FIG. 1, a light chain variable region having at least 70% identity tothe amino acid sequence of FIG. 2, or both said heavy chain variableregion and said light chain variable region.
 14. A method foridentifying a tested individual with a high probability of having amalignant disease comprising: (a) obtaining a body fluid sample fromsaid individual; (b) contacting said sample with at least one of themonoclonal antibodies of claim 1, or a monoclonal antibody, wherein saidantibody binds to an antigen to which any one of the monoclonalantibodies of claim 1 specifically bind; (c) determining the extent ofbinding of said monoclonal antibody to T-cells within said sample; and(d) comparing the extent of (c) to the extent of binding of themonoclonal to T-cells in a sample obtained from a healthy individual,wherein a significant difference between said extents of bindingindicate that said tested individual has a high probability of having amalignant disease.
 15. The method according to claim 14, wherein beforestep (b) the peripheral blood mononuclear cells are separated, saidseparated the peripheral blood mononuclear cells are then contacted instep (c) with said monoclonal antibody.
 16. The method according toclaim 14, wherein said body fluid is blood.
 17. The method according toclaim 14, wherein the extent of binding of the monoclonal antibodiesobtained from said tested individual is higher than the extent ofbinding of the same monoclonal antibody to T-cells of healthyindividuals.
 18. The method according to claim 17, wherein said specificmalignant disease is prostate carcinoma.
 19. The method according toclaim 14, wherein the extent of binding of the monoclonal antibody toT-cells obtained from said tested individual is lower than the extent ofbinding of the same monoclonal antibody to T-cells of healthyindividuals.
 20. The method according to claim 19, wherein said specificmalignant disease is breast carcinoma or colon carcinoma.
 21. Acomposition comprising at least one monoclonal antibody of claim 1together with a carrier.
 22. A kit comprising at least one monoclonalantibody of claim 1 together with a conjugate of a specific bindingpartner for said monoclonal antibody, and a label capable of producing adetectable signal.
 23. A method for the treatment of a malignant diseasecomprising administering to an individual in need a therapeuticallyeffect amount of the composition of claim
 21. 24. The method accordingto claim 23, wherein said malignant disease is a solid tumor, prostatecarcinoma, breast carcinoma, or colon carcinoma.
 25. A compositioncomprising of a therapeutically effective amount of one or more of thepeptides of claim 10, together with a carrier.
 26. A method for thetreatment of a malignant disease comprising administering to anindividual in need a therapeutically effect amount of the composition ofclaim
 25. 27. The method according to claim 26, wherein said malignantdisease is a solid tumor, prostate carcinoma, breast carcinoma, or coloncarcinoma.